Reproductive function is governed by changes in the secretory pattern of inherently pulsatile gonadotropin releasing hormone (GnRH) neurons dispersed within the hypothalamus. GnRH neurons receive inputs from other GnRH, glutamate (Glu), and GABA neurons, but the means of synchronized GnRH secretion remains enigmatic. Recently, immortalized GnRH-secreting GT1 neurons were shown to express gap junctions (GJs) and a GJ subunit protein connexin (Cx). Since GJs facilitate signaling and coordinate activity between cells, (1) we will determine the role of functional GJs in the synchronous, pulsatile pattern of basal GnRH neurosecretion. Using superfused GT1 neurons, we will analyze GnRH secretion before and after GJ blockers, and quantify functional GJs by recovery of fluorescence after photobleaching. Since Glu and GABA can affect the onset of puberty, (2) we will examine the effects of Glu and GABA on GJ assembly and Cx expression in GnRH neurons. We will assay GT1-GnRH secretion in static culture before and after Glu and GABA agents, quantify functional GJs, and evaluate Cx expression by microscopy and molecular methods. Since GJ formation may promote GnRH surges, (3) we will produce overexpression of Cxs in GnRH neurons, quantify functional GJs, and correlate their numbers with GnRH pulse characteristics. We will transfect GT1 cells with Cx cDNAs and confirm their increased expression by Northern and western blotting. We will superfuse these cells, analyze their basal and stimulated GnRH secretory profiles, quantify their functional GJs, and correlate numbers of GJs with GnRH pulse amplitude and frequency. These studies will elucidate mechanisms of coordinated GnRH neurosecretion, hypothalamic changes during puberty, and key issues regarding clinical infertility.